We propose to continue a multidisciplinary project to understand the mechanisms by which aberrant RNA and DNA structures trigger gene silencing. Two general approaches are intertwined in this work. First, we examine molecular responses following introduction of novel RNA or DNA into the organism. We propose to use multiple high resolution analyses (molecular, genetic, and biochemical) and to apply these where possible on a genomewide scale. The capability and potential impact of such analysis relies on tools and protocols developed during the previous granting period (and the subject of many of our publications during this period), including whole genome approaches to chromatin structure, transcription, steady state mRNA and small RNA patterns, and precise profiling of translation and other transient RNA dynamics. Second, we examine the natural roles of the above informational surveillance pathways in developmental and pathological contexts. Approaches parallel those above, including biochemical, molecular, and phenotypic analysis of silencing-treated molecular machinery in development and response to pathological conditions. The specific aims of the proposed research are: 1. Investigate the roles, mechanisms, and triggers of gene silencing in response to foreign/noncannonical RNA. 2. Investigate recognition and specific gene silencing in response to unusual DNA structures and sequence. 3. Investigate the interplay between RNA-triggers of gene silencing and chromatin associated nuclear events. 4. Continue to develop and refine tools and assays for studies of genetic activity and silencing in C. elegans. Studies of gene silencing have considerable potential for long term impact. First, as we understand mechanisms of gene silencing, we acquire the ability to specifically and effectively silence genes within cells or in an organism, generating a significant toolkit for functional genomic research, and aiding in the development of tools for gene-based therapeutics. Second, an understanding of gene silencing mechanisms allows improved design of systems for deliberately expressing specific genes in vivo. Such expression can provide significant advantages for investigations of biological function, for experimental elucidation of disease pathways, and for eventual intervention in biological systems (gene therapy). Third, because gene silencing mechanisms are indicative of a variety of cellular gene regulation mechanisms, work on gene silencing has provided valuable insights into normal gene regulation. Fourth, many gene silencing mechanisms reflect the response of the cell/organism to DNA or RNA that is viewed as foreign. PUBLIC HEALTH RELEVANCE: A greater understanding of signals that activate and silence regions of the genome will illuminate the fundamental mechanisms that our cells use (i) to properly control the activity of each of their genes and (ii) to protect themselves from unwanted genetic activity in the form of viruses and other genomic parasites. This research program applies a variety of information-based and experimental approaches directed toward that understanding.